djt2 at po.CWRU.Edu (Dennis J. Templeton) writes:
>In a previous article, pakapke at IASTATE.EDU () says:
>>>> We would like to get away from in vitro packaging our phage DNA and
>>simply electroporate it into the cell directly. Anyone in net-land had
>>experience doing this? Are there any references published using this
>>technique to produce a phage library?
>>
>Hmm. I don't have a direct answer for you, but I would be cautious about
>trying to electroporate DNA as large as the concatemers of lambda you get
>after ligation.
>On the other hand, we spent months making a good cDNA library out of
>LambdaZAP, THEN got experienced in electroporation of coli. Given the high
>electrocompetency rate (10e10 is not unrealistic) it seems we might have
>been just as successful at making our cDNA library in pBluescript.
>Here are some numbers:
>5 ug A+ RNA, makes about 5 ug DS cDNA
>Ligating 1 ug of this into 0.5 ug of *prepared* (see below) pBluescript
> should result in about 0.1 ug of circularized vector (probably
> more)
>Electroporation of this (in several batches) has the potential to generate
>10e8 to 10e9 clones.
>From a practical standpoint, the high electroporation efficiencies are at
>very low levels of supercoiled plasmid. We have never gotten more than
>10e6 clones from a single pulse, but 10 pulses of a ligation are practical,
>and 10e6 is plenty of cDNA's anyway.
>Input from any who has made libraries this way would be appreciated, and
>those contemplating it should look at the article in this month's
>BioTechniques (13, Nr6. 862-864, 1992) wherein the authors describe using
>pBluescript to make T-tailed vector to clone cDNA's a al Okayama and Berg.
>good luck,
>dennis
We have been trying to do the same thing recently but have been unsuccessful
at achieving any significant level of introduction of lambda DNA, even though
plasmid gives us >10e9 transformants/microgram. Anyone else have luck with
lambda DNA and electroporation?
Rex Chisholm
chisholm at worms.cmsbio.nwu.edu